Method for detecting microorganisms and microorganism detecting apparatus

ABSTRACT

It is possible to determine the presence of bacteria in a sample solution in a shorter period of time without changing a conventional incubating method. Bacteria in a sample solution are incubated in, for example, a sterilized agar medium  10  having a layer thickness of 0.1 μm to 1 μm formed on an electrode of a crystal resonator  2 , and an oscillation frequency is measured. When the bacteria proliferate, the mass of the entire crystal resonator  2  increases, and the oscillation frequency decreases. Therefore, by monitoring presence of such a change over time, presence of bacteria in the sample solution can be determined quickly.

This is a Divisional application of U.S. Ser. No. 12/931,790 filed Feb.10, 2011.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting the presence ofmicroorganisms such as bacteria and the rate of proliferation ofmicroorganisms with a piezoelectric vibrator, and to a microorganismdetecting apparatus.

2. Description of the Related Art

Nowadays, consciousness regarding food safety has been growing, andearly detection of putrefactive bacteria which deteriorates food,beverage, and the like is an important issue.

Conventionally, as means for determining whether or not putrefactivebacteria are contained in food, beverage, or the like, there have beenused measurement methods such as immuno assay, enzyme-linked immunosorbent assay (ELISA), gas chromatograph/mass spectrometry (GC/MS),liquid chromatograph/mass spectrometry (LC/MS), and the like. However,pre-treatment in these methods are complicated, and it is hard to saythat accuracy of determination is sufficient. Accordingly, a method totake long time to incubate putrefactive bacteria is common.

This incubation method is to incubate putrefactive bacteria which have apossibility to exist in a sample solution for two to four days atincubation temperatures from 30° C. to 60° C. for example, and thenperform visual examination of a colony. However, it is difficult toapply this method to a subject of examination which needs to bedetermined in a short period, such as food and beverages, and thus thereis a problem that shipment after a short period from manufacturing isnot possible.

Translated National Publication of Patent Application No. 2000-513436(claims 1, 7, and 15) describes a method for identifying a biologicalsample, and describes that components which coupled to a sensor arrayare determined directly by measuring increase in mass on a surfacethereof, that a method for detecting increase in mass on the surface isto use a crystal oscillator balance, and that the biological sample isfungi, viruses, or bacteria. However, the present invention is notdescribed in this publication.

SUMMARY OF THE INVENTION

The present invention is made in view of such situation, and it is anobject thereof to provide a method capable of detecting the presence ofmicroorganisms in a sample solution and the rate of proliferation ofmicroorganisms easily and quickly by a shorter period of time.

The present invention is a method for detecting microorganismsincluding:

using a piezoelectric vibrator having electrodes formed on both facesand supplying a culture medium layer, which is an absorption layerformed on the electrode on one face side of the piezoelectric vibrator,with a sample solution in which it is possible that microorganisms whichare detection targets are mixed; and

oscillating the piezoelectric vibrator by an oscillation circuit andmeasuring an oscillation frequency of the piezoelectric vibrator,

wherein at least one of presence of microorganisms and a rate ofproliferation of microorganisms is obtained based on a change over timein the oscillation frequency.

Further, another invention is a method for detecting microorganismsincluding:

using a piezoelectric vibrator having electrodes formed on both facesand supplying the antibody layer, which is an absorption layer formed onthe electrode on one face side of the piezoelectric vibrator, with asample solution in which it is possible that microorganisms which aredetection targets having an antigen to be absorbed in the antibody layerthrough antigen-antibody reaction are mixed;

supplying the antibody layer with a liquid culture medium; and

oscillating the piezoelectric vibrator by an oscillation circuit andmeasuring an oscillation frequency of the piezoelectric vibrator,

wherein at least one of presence of microorganisms and a rate ofproliferation of microorganisms is obtained based on a change over timein the oscillation frequency.

Further, specific examples of the detecting methods are described below.

(a) the culture medium layer is a sterilized agar medium having a layerthickness of 0.1 μm to 1 μm.

(b) the piezoelectric vibrator includes a first vibration area fordetecting microorganisms structured by forming electrodes on both facesof the piezoelectric vibrator, and a second vibration area for referenceprovided in an area different from the first vibration area via anelastic boundary layer and structured by forming electrodes on bothfaces of the piezoelectric vibrator, and the absorption layer is formedon the electrode on one face side of the first vibration area, but isnot formed on either of the electrodes of the second vibration area.

(c) there is included displaying measurement values of the oscillationfrequency as chronological data on a display unit.

(d) the measuring of a change over time in the oscillation frequency isperformed in a state that a piezoelectric vibrator is placed in anincubation container at a constant temperature including a temperatureadjusting part.

(e) the microorganisms are bacteria and fungi.

Still another invention is a detecting apparatus using a piezoelectricvibrator having electrodes formed on both faces, allowing microorganismswhich are detection targets to be absorbed in an antibody layer formedon the electrode on one face side of the piezoelectric vibrator, anddetecting detection targets by obtaining at least one of presence ofmicroorganisms and a rate of proliferation of microorganisms based on achange over time in an oscillation frequency obtained from thepiezoelectric vibrator, the detecting apparatus including:

an incubation container including an incubation space, to which a samplesolution is supplied, for retaining a piezoelectric vibrator with a faceon which the antibody layer is formed is directed to the incubationspace;

a sample solution supply part supplying the incubation container with asample solution in which it is possible that microorganisms which aredetection targets having an antigen to be absorbed in the antibody layerthrough antigen-antibody reaction are mixed;

a culture medium supply part supplying the incubation container with aliquid culture medium; and

an oscillation circuit for oscillating the piezoelectric vibrator.

Specific examples of the detecting methods are described below.

(f) the piezoelectric vibrator includes a first vibration area fordetecting microorganisms structured by forming electrodes on both facesof the piezoelectric vibrator with the absorption layer being formed onthe electrode on one face side thereof, and a second vibration area forreference provided in an area different from the first vibration areavia an elastic boundary layer and structured by forming electrodes onboth faces of the piezoelectric vibrator, where the absorption layer isnot formed on either of the electrodes on the both faces, and

wherein the detecting apparatus includes a first oscillation circuitoscillating the first vibration area and a second oscillation circuitoscillating the second oscillating area.

(g) the incubation container includes a temperature adjusting part formaintaining an incubation space at a constant temperature.

(h) the microorganisms are bacteria and fungi.

In the present invention, microorganisms in a sample solution isincubated in a culture medium formed on an electrode of a piezoelectricresonator, and proliferation of microorganisms is detected as a changein resonance frequency. Consequently, the presence of microorganisms andthe rate of proliferation of microorganisms can be detected simply andquickly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an external perspective view partially illustrating anincubation apparatus according to an embodiment of the presentinvention;

FIG. 2 is an exploded perspective view illustrating upper face sides ofrespective parts of the incubation apparatus;

FIG. 3 is a vertical cross-sectional view illustrating the incubationapparatus;

FIG. 4 is a vertical cross-sectional view schematically illustrating acrystal resonator on which a culture medium layer is formed on an upperface of an electrode, according to the embodiment of the presentinvention;

FIG. 5 is a vertical cross-sectional view schematically illustrating thecrystal resonator in a state that microorganisms are incubated on aculture medium layer to which a sample solution is added;

FIG. 6 is a diagram schematically illustrating the overall structure ofthe incubation apparatus;

FIG. 7 is a characteristic chart illustrating an example of a frequencytemperature characteristic of the crystal resonator;

FIG. 8 is a characteristic chart illustrating an example of ameasurement result by the incubation apparatus;

FIG. 9 is an explanatory diagram illustrating the structure of amicroorganism detecting apparatus according to a second embodiment;

FIG. 10 is a schematic view illustrating the structure of a crystalresonator disposed in an incubator of the microorganism detectingapparatus;

FIG. 11 are plan views illustrating the crystal resonator and the wiringsubstrate which are used in the microorganism detecting apparatus;

FIG. 12 is a perspective view illustrating a piezoelectric sensorincluding the crystal resonator; and

FIG. 13 are characteristic charts illustrating examples of measurementresults by the microorganism detecting apparatus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An embodiment of a method for detecting microorganisms according to thepresent invention will be described with reference to the drawings. FIG.1 and FIG. 2 are an external perspective view and an explodedperspective view, respectively, which partially illustrate an incubationapparatus used in this embodiment. Numeral 7 denotes an incubator(incubation container) which is formed of a support body 71 and a cover81, and is structured of a sealing member 3A, a wiring substrate 3, acrystal resonator 2, and a crystal pressing member 4 which are stackedfrom a lower side in this order between the support body and the cover,as illustrated in FIG. 3.

The crystal resonator 2 which is a piezoelectric resonator is formed ofa circular-shaped crystal piece 21, which is a piezoelectric piece, anexcitation electrode 22, and lead-out electrodes 24, 25 (not illustratedin FIG. 4) as illustrated in FIG. 2. On a front face side of the crystalpiece 21, the excitation electrode 22 in a foil shape is formed in acircular shape smaller in diameter than the crystal piece 21, and oneend side of the lead-out electrode 24 in a foil shape is formed to beconnected to the excitation electrode 22. This lead-out electrode 24 isbent along an end face of the crystal piece 21 and is routed to a rearface side of the crystal piece 21. On the other hand, also on the rearface side of the crystal piece 21, the excitation electrode 22 and thelead-out electrode 25 are formed to be connected in the same layout asthe front face side. An equivalent thickness of the excitation electrode22 and the lead-out electrodes 24, 25 is, for example, 0.2 μm, and gold,silver, or the like for example is used as a material for theelectrodes.

As illustrated in FIG. 4, on the excitation electrode 22 on the surfaceside (side to be in contact with a sample solution) provided on thecrystal piece 21, there is formed a culture medium layer 10 which is anabsorption layer to be an incubating environment for microorganisms, forexample, bacteria such as putrefactive bacteria. As this culture mediumlayer 10, for example, a sterilized agar medium can be used. A layerthickness of this agar medium is, for example, 0.1 μm to 1 μm. This isbecause when the layer thickness is 0.1 μm or less, incubation ofmicroorganisms is not possible, and when the layer thickness is morethan 1 μm, the crystal resonator is not driven easily because the agarhas high viscosity. A method for forming the culture medium layer 10 onthe excitation electrode 22 is performed as follows for example. First,on both the front and rear faces of a crystal wafer, electrode patternsare formed at positions corresponding to the positions of formingnumerous crystal pieces. Then, on a rotatable vacuum chuck (rotationstage including a vacuum absorbing function), this crystal wafer is heldhorizontally in a state that the center of the wafer and the center ofrotation are aligned. A solution containing a culture medium which isadjusted to a predetermined agar concentration is supplied to the centerof the wafer, and the spin chuck is rotated by a motor coupled to arotation shaft of the spin chuck, thereby spreading the solution on thesurface of the wafer to form a thin liquid film. The thickness of thisliquid film can be adjusted according to the rotation speed, and thus byadjusting this rotation speed, the culture medium layer 10 having alayer thickness of 0.1 μm to 1 μm can be formed on the wafer.Thereafter, the wafer is divided by dicing to obtain individual piecesof crystal resonators. Although the culture medium layer 10 is drawn tobe formed only on the excitation electrode in FIG. 4, the culture medium10 is formed on the entire surface of the crystal piece by this spincoating method. Also in this case, by structuring such that the ratio ofthe area of the electrode to the area of the entire surface of thecrystal piece is larger, the weight of the culture medium would notaffect driving of the crystal resonator. An oscillation frequency ofthis crystal resonator at this point is assumed as a reference fordetecting microorganisms. At this time, it is assumed that when the agarconcentration is constant, viscosity resistance thereof is constant, andthe Kanazawa-Gordon formula is used.

Referring back to FIG. 2, the wiring substrate 3 is formed of a printedcircuit board for example, and electrodes 31, 32 are provided at aninterval on a surface thereof. A through hole 33 for a recessed portionforming an air-tight space, in which the excitation electrode 22 on therear face side of the crystal resonator 2 is exposed as will bedescribed later, is formed between the electrodes 31, 32, and thethrough hole has a bore formed to have a size in which the excitationelectrode 22 can be accommodated. On a rear end side of the wiringsubstrate 3, connection terminal parts 34, 35 are provided and connectedelectrically to the electrodes 31, 32, respectively, via conductivepaths. The sealing member 3A is formed of a circular body with arecessed portion formed in a center portion, and has a role to block alower face of the through hole 33 and form an air-tight space which is arear side atmosphere of the crystal resonator 2. The lead-out electrodes24, 25 of the crystal resonator 2 are connected electrically by aconductive adhesive.

The crystal pressing member 4 is made in a shape corresponding to thewiring substrate 3 using an elastic material, for example a siliconerubber. A lower face of the crystal pressing member 4 is, as illustratedin FIG. 3, formed so as to press a peripheral portion of the excitationelectrode of the crystal resonator 2 against the support body 71 side.The crystal pressing member 4 has a role of pressing the crystalresonator 2 against an outside area of the through hole 33 formed in thewiring substrate 3, so that when the cover 81 is fitted with the supportbody 71, the crystal pressing member 4 presses the crystal resonator 2and the wiring substrate 3 against the support body 71 side.

The cover 81 is positioned onto the support body 71 by engagement ofrecessed portions 82 formed in a lower face thereof and projections 75formed on the support body 71 side as illustrated in FIG. 3, and isfixed by screws 83 which are screwed into holes 74 on the support body71 side. A heater 60 which is a temperature adjusting part is providedin the cover 81 and the support body 71 which form the incubator 7, andby this heater 60 the atmosphere in which the culture medium layer isplaced is set to an atmosphere at a preset temperature (constanttemperature atmosphere).

In the support body 71, a recessed portion 72 in which the wiringsubstrate 3 is accommodated and retained is provided, and in thisrecessed portion 72, engaging projections 73 extend in a verticaldirection and engage with engaging holes 37 a, 37 b of the wiringsubstrate 3 and engaging holes 46 a, 46 b of the crystal pressing member4, thereby fixing positions of the wiring substrate 3 and the crystalpressing member 4.

Next, a circuit part and a signal processing part of the incubationapparatus will be described. In FIG. 6, numeral 50 denotes anoscillation circuit, numeral 51 denotes a signal processing part, andnumeral 52 denotes a personal computer. A screen of this personalcomputer 52 constitutes a display unit.

The oscillation circuit 50 is connected electrically to the electrodes34, 35 formed on the wiring substrate 3, and is provided on the supportbody 71 for example. The signal processing part 51 performsanalog/digital conversion (A/D conversion) of a signal having afrequency from the oscillation circuit 50, and performs predeterminedsignal processing thereon to measure the frequency of the frequencysignal.

Next, a process of detecting whether target bacteria are present in asample solution or not using the thus structured incubation apparatuswill be described. In this example, assuming that the bacteria areputrefactive bacteria, a filter which allows passing of the putrefactivebacteria is prepared, and a sample solution is made to pass through thisfilter. Then, the passed liquid is sampled with a dropper for example,dropped onto a surface of the culture medium layer 10 on thealready-described crystal resonator 2 fitted in the support body 71, andspread thinly thereon. Thereafter, the cover 81 is fitted onto thesupport body 71, and the temperature in the incubator 7 (the temperatureof the atmosphere in which the crystal resonator 2 is placed) is raisedby the heater 60 to, for example, between 40° C. to 60° C., 45° C. inthis example.

Then the aforementioned atmosphere is kept airtight, and the samplesolution on the culture medium layer 10 evaporates, thereby turning thisatmosphere to a saturated steam atmosphere for example. On the otherhand, measurement values of the oscillation frequency of the crystalresonator 2 are taken into the personal computer 52 chronologically, andthese chronological data are displayed on the screen thereof. In theincubator 7, the total mass of the sample solution on the electrodes ofthe crystal resonator 2 and the culture medium layer 10 varies untilwater in the sample solution evaporates and saturates the atmosphere.However, once the saturated atmosphere is established, the total massbecomes table. Now, if putrefactive bacteria (detection targets 15)exist in the sample solution applied on the culture medium layer 10 asillustrated in FIG. 5, these bacteria take in nutriments in the culturemedium layer and proliferate. Thus, the mass of a portion on theelectrodes increases from the point when the proliferation starts, andthe oscillation frequency decreases. Accordingly, when it is configuredto display the chronological data of the oscillation frequency on thescreen of the personal computer 52 in the form of a graph for example,it is possible to allow recognition of existence of the putrefactivebacteria by, for example, visually detecting a rising of the oscillationfrequency.

Further, by obtaining the degree of decrease in oscillation frequency,the rate of proliferation of the putrefactive bacteria can be obtained.In this case, when the relation between the oscillation frequency andthe mass of an object mounted on the electrode 22 of the crystalresonator 2 is obtained in advance, the rates of proliferation atpredetermined time intervals can be obtained for example. Moreover, anair-pipe (air passage) and an exhaust pipe (exhaust passage) (notillustrated) which each communicate with the atmosphere are connected tothe incubator 7, and air, for example dry air, is supplied in a statethat the exhaust pipe is open, to thereby remove water on the crystalresonator 2 to some degree. Then, measurement of the oscillationfrequency may be performed after air containing, for example, slightlyless steam than the saturated steam atmosphere is sent in at the settemperature in the incubator 7, respective valves provided on theair-pipe and the exhaust pipe are closed, and water on the crystalresonator 2 evaporates slightly to cause the atmosphere to be saturatedsteam atmosphere.

In addition, it is preferred that the set value of the incubationtemperature be a temperature at which a temperature characteristic ofthe frequency of the crystal resonator becomes most stable within therange of growth temperature of bacteria. FIG. 7 illustrates an exampleof a frequency temperature characteristic of an AT-cut crystalresonator. In this case, the temperature at which a frequency changeamount per unit temperature change in the growth temperature range issmallest is 45° C., which is the temperature at the extremal value ofthe graph, and it is preferred that the incubation temperature is set tothis temperature. For beverages, a sample solution can be prepared byjust passing through the filter, but when the presence of putrefactivebacteria in food is checked, a sample solution can be prepared byincubating the putrefactive bacteria by a method based on an officialmethod or by mixing the putrefactive bacteria directly with water andthen passing this water through the filter.

In such a method for detecting bacteria, after a sample solution isdropped onto the culture medium layer 10 and the frequency becomesstable, the oscillation frequency starts to decrease for example after alapse of a few tens of minutes. Thus, the presence of bacteria can bedetected by a short period of time, and hence whether the putrefactivebacteria are present in food or beverage can be detected quickly.Further, the operation is simple and the accuracy of measurement ishigh.

Note that in the present invention, microorganisms include bacteriafungi, and the like.

Using the above-described incubation apparatus, the sample solutioncontaining putrefactive bacteria was adhered on the culture medium layer10 of the crystal resonator 2 as already described, and after theoscillation frequency becomes table, a change over time of a frequencydifference with respect to this frequency was measured. A measurementresult is illustrated in FIG. 8. As the crystal resonator, an AT-cutcrystal resonator having a diameter φ of 8.7 mm of 9 MHz (with anelectrode diameter φ of 5.0 mm) is used, and measurement of frequency isperformed at 10 mHz/sec. As a strain to be a detection target, astandard strain (ATCC49025) of Alicyclobacillus acidoterrestris is used,and this strain is incubated at 45 degrees Celsius in an agar medium onthe crystal resonator with a layer thickness of 0.5 μm. Here, an yeastextract, glucose, several trace elements, and so on are added to thisagar. As illustrated in FIG. 8, the frequency decreases by 1 Hz afterabout 0.5 hour from start of incubation, and the inherent frequencykeeps decreasing thereafter. This is due to increase in mass caused byproliferation of the putrefactive bacteria, and from this increase,proliferation of the putrefactive bacteria can be confirmed. Here, as adevice for detecting the frequency, a device sold by the applicant(NAPiCOS system, registered trademark) capable of measuring thefrequency with quite high accuracy is used.

Further, by obtaining an analytical curve representing a relationbetween the weight and the oscillation frequency in advance, a frequencydifference can be converted into a mass increase amount. Thus, the rateof change in mass at each incubation elapsed time, that is, the rate ofproliferation of the detection target at each incubation elapsed timecan be digitized.

Next, a second embodiment of detecting microorganisms such as bacteriausing an antibody will be described. A method for detectingmicroorganisms according to this example is different from the detectingmethod according to the first embodiment, in which the culture mediumlayer 10 is formed on the excitation electrode 22 to perform incubation,in that an antibody layer as an absorption layer is provided on theexcitation electrode 22 of a crystal resonator 2 a, and a culture mediumsolution (liquid culture medium) is supplied to the atmosphere in whichthis crystal resonator 2 a is mounted, to thereby perform incubation ofmicroorganisms. FIG. 9 illustrates the structure of a microorganismdetecting apparatus 70 using the method for detecting microorganismsaccording to the second embodiment. In FIG. 9, components common to thefirst embodiment are given the same reference numerals as those in FIG.1 to FIG. 6.

In the microorganism detecting apparatus 70 illustrated in FIG. 9, anincubator 700 is in common with the incubator 7 illustrated in FIG. 2and FIG. 3 in that the crystal pressing member 4 and the sealing member3A are used to retain the piezoelectric sensor constituted of thecrystal resonator 2 a and a wiring substrate 3 a in the incubator 700.On the other hand, the incubator is different from the incubator 7according to the first embodiment, in which a sample solution is appliedon the culture medium layer 10 and this layer is disposed in a closedatmosphere, in that a supply port 704 for supplying a sample solutioninto the incubator 700 and a discharge port 705 for discharging a samplesolution from inside the incubator 700 are provided on the cover 81 ofthe incubator 700, and a sample solution or culture medium solution canbe supplied externally.

The supply port 704 of the incubator 700 is connected to a supply liquidswitching part 703 including a not-illustrated sample loop which retainsa sample solution and a culture medium solution temporarily whilesuppressing mixing of them and switching a connection destination of thesupply port 704 between a culture medium solution supply part 701constituted of a syringe pump retaining the culture medium solution anda sample solution supply part 702 constituted of a syringe retaining thesample solution, and so on. On the other hand, the discharge port 705 isconnected to a discharged liquid receiving tank 708 via a suction pump707, and this suction pump 707 is used to supply the culture mediumsolution and the sample solution to the incubator 700 and to dischargethem therefrom.

Further, the incubator 700 according to this example is different fromthe first embodiment performing temperature adjustment using the heater60 in that temperature adjustment of the atmosphere in which the crystalresonator 2 a is disposed is performed by disposing the incubator in,for example, a hot-air type constant temperature chamber 706 togetherwith the oscillation circuit 50 connected to the crystal resonator 2 a.

Further, the point that the incubator is connected to the oscillationcircuit 50 for the crystal resonator 2 a and is connected to thepersonal computer 52 via the signal processing part 51 is the same as inthe first embodiment illustrated in FIG. 6.

Here, in the crystal resonator 2 a according to this embodiment, twopairs of excitation electrodes 22 a, 22 b are provided on a front andrear faces of a crystal piece 21 as illustrated in FIG. 10, FIG. 11( a),and FIG. 11( b), and form a first vibration area 20 a and a secondvibration area 20 b, respectively. On an upper face of the excitationelectrode 22 a on a front face side of the first vibration area 20 a,there is formed an antibody layer 11 carrying an antibody assuming amembrane protein contained specifically in a surface of bacteria, whichare the detection targets 15, as an antigen and reacting with thisantigen, and the antibody layer forms a vibration area for detectingmicroorganisms where bacteria are made to be absorbed into the surfaceof the excitation electrode 22 a. On the other hand, on the surface ofthe excitation electrode 22 b of the second vibration region 20 b, ablocking layer 12 constituted of a protein or the like which does noteasily react with the antibody is formed, and the bacteria are noteasily absorbed into the surface of the excitation electrode 22 b.Accordingly, the second vibration area 20 b forms a vibration area forreference to detect a change in oscillation frequency due to any otherfactor (for example, an environmental change which will be describedlater) which is not due to absorption of microorganisms.

Regarding fixing of the antibody to the excitation electrode 22 a, forexample, a reagent containing the antibody is supplied to an upper faceside of the crystal resonator 2 a on which the other excitationelectrode 22 b is masked in advance, to thereby form the antibody layer11 on a surface of the exposed excitation electrode 22 a. Formation ofthe blocking layer 12 on the other excitation electrode 22 b issimilarly performed by, for example, masking the excitation electrode 22a on which the antibody layer 11 is to be formed, and then making theother excitation electrode 22 b to be in contact with a reagentcontaining a protein to be the blocking layer.

As illustrated in FIG. 12, the crystal resonator 2 a is disposed on thewiring substrate 3 a capable of taking out oscillation frequenciesseparately from the first vibration area 20 a and the second vibrationarea 20 b, and the areas 20 a, 20 b are connected to separateoscillation circuits 50. In the example illustrated in FIG. 11 and FIG.12, the excitation electrodes 22 a, 22 b on the front face side are ledto a connection terminal part 35 via a common lead-out electrode 24 andan electrode 32 on the wiring substrate 3 side, and is connected to aground line on the oscillation circuit 50 side. On the other hand, theexcitation electrodes 22 a, 22 b on the rear face side are led toconnection terminal parts 34 via separate lead-out electrodes 25 andelectrodes 31 on the wiring substrate 3 side, and are connected to therespective independent oscillation circuits 50.

Spore forming bacteria, for example Clostridium perfringens, are able toproliferate under a relatively high temperature condition of, forexample, 50° C. to 70° C., and become deterioration factors in foodstored under such a temperature condition. Accordingly, when the sporeforming bacteria are the detection targets 15, it is necessary to retainthe crystal resonator 2 a contacted with the sample solution under thesame temperature condition as the storing condition of food, and measurethe presence of bacteria to be absorbed in the antibody layer 11 and thepresence of proliferation of bacteria. On the other hand, it is knownthat the oscillation frequency characteristic of the crystal resonator 2a changes according to a change in the ambient temperature, a change inviscosity of fluid, and the like (hereinafter referred to asenvironmental changes for convenience). However, it is difficult todiscriminate by measurement in a short time whether a change inoscillation frequency is due to absorption of bacteria into the antibodylayer 11 or due to a variation in temperature control and a change inviscosity of the surrounding fluid in the constant temperature chamber706.

Accordingly, in the microorganism detecting apparatus 70 according tothis embodiment, the first vibration area 20 a provided with theantibody layer 11 which absorbs bacteria and the second vibration area20 b provided with the blocking layer 12 which does not absorb bacteriaare disposed under the same environment. Thus, changes in oscillationfrequency due to both the absorption of bacteria and the environmentalchanges are obtained from the first vibration area 20 a, whereas changesin oscillation frequency only due to surrounding environmental changesand excluding absorption of bacteria can be obtained from the secondvibration area 20 b. Thus, by taking a difference between oscillationfrequencies obtained respectively from the first and second vibrationareas 20 a, 20 b, the influence of the environmental changes is removedfrom the oscillation frequency of the first vibration area 20 a, andonly the change in the oscillation frequency due to absorption ofbacteria can be taken out.

Hereinafter, operation of the above-described microorganism detectingapparatus 70 will be described. First, a piezoelectric sensor isattached in the incubator 700, and the temperature in the constanttemperature chamber 706 is set so that the ambient temperature of theincubator 700 and the oscillation circuit 50 becomes the storagetemperature of food contained in a sample solution for example. When thetemperature of the constant temperature chamber 706 becomes stable, thesupply liquid switching part 703 is connected to the culture mediumsolution supply part 701 side and the suction pump 707 is activated, anda culture medium solution is filled as a supplied liquid buffer in theincubator 700 and pipes connected thereto. Then the oscillation circuit50 is activated to start obtaining oscillation frequencies from thefirst, second vibration areas 20 a, 20 b.

Thereafter, the connection destination of the supply liquid switchingpart 703 is switched to the sample solution supply part 702 to supplythe sample solution to the incubator 700, and the suction pump 707 isstopped once at the timing the inside of the incubator 700 is replacedwith the sample solution. At this time, if the sample solution containsputrefactive bacteria such as spore-forming bacteria which undergoantigen-antibody reaction with the antibody of the antibody layer 11,the putrefactive bacteria are absorbed into the antibody layer 11, andthe oscillation frequency of the first vibration area 20 a lowers asillustrated in the period described as “putrefactive bacteria absorbed”in FIG. 13( a) and FIG. 13( b).

On the other hand, on the second vibration area 20 b side where theblocking layer 12 is provided, absorption of the putrefactive bacteriahardly occurs, and thus there occurs no change in oscillation frequencyin the aforementioned period. Here, for simplicity, in the examplesillustrated in FIG. 13( a) and FIG. 13( b), the culture medium solutionsupplied from the culture medium solution supply part 701 and the samplesolution supplied from the sample solution supply part 702 are adjustedin advance to almost the same temperature as the temperature in theincubator 700 which is adjusted in the constant temperature chamber 706.Further, it is assumed that the culture medium solution and the samplesolution have substantially the same viscosity, and no change inoscillation frequency occurs due to switching between the culture mediumsolution and the sample solution.

Thus, when a predetermined time has passed and a time by which asufficient amount of microorganisms can be absorbed in the antibodylayer 11 has passed, the connection target of the supply liquidswitching part 703 is switched again to the culture medium solutionsupply part 701, and then the suction pump 707 is activated so as toreplace the inside of the incubator 700 with the culture mediumsolution. When the inside of the incubator 700 is thus brought to astate of being filled with the culture medium solution, the suction pump707 is stopped again, and the inside of the incubator 700 is brought toa stationary state. Here, when the putrefactive bacteria absorbed intothe antibody layer 10 are alive, the putrefactive bacteria receivenutriments from the culture medium solution and proliferate. Theproliferation of the putrefactive bacteria at this time occurs near thesurface of the antibody layer 11 where these putrefactive bacteria areabsorbed. Thus, when the inside of the incubator 700 is in a stationarystate, the proliferated putrefactive bacteria spread in the culturemedium solution, and a part thereof is absorbed in the antibody layer11.

As a result, as illustrated in the period described as “putrefactivebacteria proliferate” in FIG. 13( a), the oscillation frequency on thefirst vibration area 20 a side decreases over time due to absorption ofthe proliferated putrefactive bacteria. On the other hand, on the secondvibration area 20 b side provided with the blocking layer 12, there isstill almost no decrease in oscillation frequency. In these periodsdescribed as “putrefactive bacteria absorbed” and “putrefactive bacteriaproliferate” in FIG. 13( a), when environmental changes, such astemperature changes and/or viscosity changes, occur inside the incubator700, changes in oscillation frequency accompanying these environmentalchanges occur at substantially the same widths in the first, secondvibration areas 20 a, 20 b placed at the common temperature and in thecommon culture medium solution atmosphere.

Accordingly, by taking the absolute value of the difference between theoscillation frequencies obtained from the first, second vibration areas20 a, 20 b, the changes in oscillation frequency accompanying theenvironmental changes are cancelled out, and a change in oscillationfrequency only due to absorption of the putrefactive bacteria can betaken out. Then, similarly to the first embodiment, the amount ofabsorbed putrefactive bacteria is recognized by using an analyticalcurve or the like indicating the relation between the weight of theputrefactive bacteria obtained in advance and the amount of decrease inoscillation frequency.

There may be a case where the amounts of changes in the oscillationfrequencies of the first, second vibration areas 20 a, 20 b due to theenvironmental changes are not strictly equal. In this case, variationsin the oscillation frequencies accompanying such environmental changescan be cancelled out properly by multiplying each of the oscillationfrequencies with a proportionality constant obtained in advance to makethe amounts of changes in oscillation frequency per unit change oftemperature, viscosity, and so on, to be close to each other, and thentaking the difference between both the oscillation frequencies.

In addition, when the proliferation of the putrefactive bacteriaproceeds exponentially, the concentration of putrefactive bacteria inthe culture medium solution increases, precipitation of the putrefactivebacteria on the blocking layer 12 or the like occurs, and theputrefactive bacteria precipitated on the second vibration area 20 balso begins to proliferate. As a result, as illustrated in the perioddescribed as “proliferate also in the second vibration area 20 b” inFIG. 13( a), there may be a case where the oscillation frequency on thesecond vibration area 20 b side begins to decrease. However, as alreadydescribed, the oscillation frequency on the second vibration area 20 bside barely changes in the period until proliferation of theputrefactive bacteria is observed in this area 20 b (equivalent to theperiods “putrefactive bacteria absorbed” and “putrefactive bacteriaproliferate” in FIG. 13( a)). Therefore, measurement of the presence ofputrefactive bacteria and the rate of proliferation can be performedproperly by using data obtained in these periods.

When the putrefactive bacteria contained in the sample solution aredead, the oscillation frequency of the first vibration area 20 adecreases in the period described as “putrefactive bacteria absorbed” asillustrated in FIG. 13( b). However, these putrefactive bacteria do notproliferate, and thus the oscillation frequency of the first vibrationarea 20 a does not change any further. Accompanying that theputrefactive bacteria are hardly absorbed, the oscillation frequency ofthe second vibration area 2 b stays at a constant value. Also in such acase, a change in oscillation frequency due to environmental changes canbe cancelled out by taking the absolute value of the difference inoscillation frequencies obtained from the first, second vibration areas20 a, 20 b.

In the microorganism detecting apparatus 70 according to the secondembodiment, the crystal resonator 2 a provided with the antibody layer11 is disposed in the incubator 700, the sample solution is supplied tothis incubator 700, and the presence of microorganisms which are thedetection targets and the rate of proliferation of microorganisms aredetected based on a change in oscillation frequency of the crystalresonator 2 provided with the antibody layer 11. By conventionallyperformed approaches to observe microorganisms such as bacteria whichare made to proliferate in an agar medium with a microscope or the like,a test period of about three days to one week for example is requiredfor finding the presence of microorganisms and the rate ofproliferation. In this aspect, a method for detecting microorganismsusing a piezoelectric vibrator such as the crystal resonator 2 a hashigh sensitivity, and is capable of detecting a mass change of a quitesmall amount, such as nanograms. Accordingly, it is not necessary towait for proliferation of microorganisms until it can be observedoptically, and it becomes possible to detect the presence of thedetection targets and the rate of proliferation by a much shorter periodof time than by conventional methods, such as 10 hours to two days.

Further, in this method using the crystal resonator 2 a with highsensitivity, the influence of environmental changes, such as a change inambient temperature of the crystal resonator 2 a and changes intemperature and viscosity of liquid (sample solution or culture mediumsolution) in contact with the crystal resonator 2 a, on the oscillationfrequency of the crystal resonator 2 a becomes large. Here, themicroorganism detecting apparatus 70 according to the second embodimentuses the crystal resonator 2 a having the first vibration area 20 a fordetecting microorganisms which is provided with the antibody layer 11for absorbing microorganisms, and the second vibration area 20 b forreference which is not allowed to absorb microorganisms. Accordingly, bytaking the difference in oscillation frequencies obtained from both thevibration areas 20 a, 20 b, it is possible to cancel out the influenceof the environmental changes, thereby allowing to properly grasp achange in oscillation frequency due to absorption of microorganisms.

Here, it is possible to replace the components of the incubatingapparatus described in the first embodiment and the microorganismdetecting apparatus 70 described in the second embodiment appropriatelyas necessary. For example, the heater 60 used in the first embodimentmay be provided in the microorganism detecting apparatus 70 of thesecond embodiment, or conversely the incubator 7 of the first embodimentmay be disposed in the constant temperature chamber 706. Moreover, themicroorganism detecting apparatus 70 of the second embodiment, which isof what is called flow injection type, may be structured as a batch typesimilarly to the incubator 7 according to the first embodiment. In thiscase, for example, the crystal resonator 2 a on which a sample solutionis applied in advance is disposed in the antibody layer 11 in theincubator 700 which is not provided with the supply port 704 and thedischarge port 705, the culture medium solution is supplied thereafterto the inside of the incubator 700 and then the support body 71 iscovered with the cover 81, and the inside of the incubator 700 isadjusted at a preset temperature, thereby performing detection ofmicroorganisms. Besides this, a piezoelectric sensor (hereinafterreferred to as a twin sensor) using the crystal resonator 2 a having thefirst vibration area 20 a for detecting microorganisms and the secondvibration area 20 b for reference may be used in the incubationapparatus according to the first embodiment. Conversely, when theinfluence of environmental changes would not be a problem, the crystalresonator 2 described in FIG. 2 in which the vibration area 20 b forreference is not provided may be used instead of the twin sensor in themicroorganism detecting apparatus 70 according to the second embodiment.

What is claimed is:
 1. A method for detecting microorganisms,comprising: providing a piezoelectric vibrator having electrodes formedon both faces; supplying a culture medium layer, which is an absorptionlayer, on the electrode on one face side of the piezoelectric vibrator,wherein the culture medium layer comprises a sterilized agar mediumhaving a layer thickness of 0.1 μm to 1 μm, and a sample solution inwhich microorganisms which are detection targets are mixed; andoscillating the piezoelectric vibrator by an oscillation circuit andmeasuring an oscillation frequency of the piezoelectric vibrator causinga proliferation of said microorganisms, detecting a rate ofproliferation of microorganisms is obtained based on a change over timein the oscillation frequency caused by a mass increase of themicroorganisms proliferating in the absorption layer during saidoscillating.
 2. The method for detecting microorganisms according toclaim 1, wherein the piezoelectric vibrator comprises a first vibrationarea for detecting microorganisms structured by forming electrodes onboth faces of the piezoelectric vibrator, and a second vibration areafor reference provided in an area different from the first vibrationarea via an elastic boundary layer and structured by forming electrodeson both faces of the piezoelectric vibrator, and the absorption layer isformed on the electrode on one face side of the first vibration area,but is not formed on either of the electrodes of the second vibrationarea.
 3. The method for detecting microorganisms according to claim 1,further comprising: displaying measurement values of the oscillationfrequency as chronological data on a display unit.
 4. The method fordetecting microorganisms according to claim 1, wherein the measuring ofa change over time in the oscillation frequency is performed in a statethat a piezoelectric vibrator is placed in an incubation container at aconstant temperature including a temperature adjusting part.
 5. Themethod for detecting microorganisms according to claim 1, wherein themicroorganisms are bacteria and fungi.
 6. A method for detectingmicroorganisms, comprising: applying a sterilized agar medium having alayer thickness of 0.1 μm to 1 μm to an electrode; after said applying,supplying a sample solution, in which it is possible that microorganismswhich are detection targets are mixed, to the agar medium so as to forman absorption layer on the agar medium on the electrode; after saidsupplying, proliferating microorganisms in said absorption layer whilethe absorption layer and agar medium are on said electrode at one faceof a piezoelectric vibrator having electrodes formed on two faces; andoscillating the piezoelectric vibrator by an oscillation circuit andmeasuring an oscillation frequency of the piezoelectric vibrator; anddetermining a rate of proliferation of the microorganisms based on achange over time in the oscillation frequency, as caused by a massincrease of the microorganisms proliferating in the absorption layerduring said oscillating.